Method for obtaining plasmid-dna by means of an aqueous biphasic system

ABSTRACT

The invention relates to a process for isolation of plasmid DNA from biomass by means of an aqueous 2-phase system having a polymer component and a salt component, characterized in that the resuspension of the biomass employed, the alkaline lysis of the biomass, the neutralization of the alkaline lysis batch and the separation of the plasmid DNA from the contaminants (such as e.g. cell debris, RNA and gDNA) are carried out in a single reaction vessel (one-pot process). According to the invention, this is rendered possible in that the neutralization of the alkaline lysis batch is carried out in one and the same container by addition of potassium phosphate and one component of the aqueous 2-phase system is therefore already present, and in that the second component of the aqueous 2-phase system is a PEG having a molecular weight of the mathematical average of about 600 g/mol to 1,000 g/mol, but is preferably formed from a mixture of PEG 600 and PEG 1000.

The invention relates to a simplified process, which is shorter in time,for isolation of plasmid DNA from biomass, such as e.g. bacteria, bymeans of an aqueous 2-phase system having a polymer component and a saltcomponent, and to the use of the plasmid DNA obtained in this way ingene therapy and in genetic vaccination. The invention furthermorerelates to a kit for carrying out the process.

One of the possibilities of isolating plasmid DNA from biomass is lysisof the biomass and subsequent separation of the clarified lysates fromthe biomass in an aqueous 2-phase system. The Dutch microbiologistBeijerinck already discovered in 1896 that after mixing agar and solublestarch in water, two aqueous phases form after some time. This propertyof some polymers was rediscovered and pursued by Per Albertson in about1960. He recognized the potential of such a system for purification ofviruses, cells and also cell constituents. Due to the high water contentof the biocompatible medium and the stabilizing properties of the phasecomponents, such phase systems are correspondingly particularly suitablefor purification of sensitive biomolecules. Aqueous 2-phase systems areobtained either by addition of two different polymers (polymer/polymersystem; e.g. polyethylene glycol (PEG) and dextran), or by a polymer(e.g. polyethylene glycol) and a highly concentrated salt (e.g.citrates, sulphates or phosphates).

The widely used techniques for the preparation of recombinant DNA andthe ever increasing interest in gene therapy for treatment of the mostdiverse diseases and genetic vaccination have caused the demand for aprocess for the purification of plasmid DNA which can also be used on anindustrial scale to increase. Alternative methods, such ascentrifugation in a caesium chloride (CsCl) density gradient or phenolextraction, are thus inconceivable for a plasmid DNA obtained fortherapeutic use because of the toxicity of the substances, in the sameway as the amount of toxic or combustible substances to be employed fora production on an industrial scale.

The prior art also includes processes for isolation of plasmid DNA inwhich aqueous 2-phase systems having a salt component and a polymercomponent are used. Cole (Biotechniques. July 1991;11(1):18, 20, 22-24)published an aqueous 2-phase system for isolation of plasmids using PEGas the polymer component and e.g. ammonium sulphate or sodium dihydrogenphosphate as the salt component. The isolation of the plasmid pTZ 18Ufrom E. coli DH5a is described concretely. The corresponding cell pelletwas broken down by means of a lysis buffer comprising SDS and NaOH. Thelysate was introduced into an appropriate phase-forming system and themixture was mixed and then centrifuged. This was repeated twice morewith the lower phase and a fresh upper phase containing no lysate. Thelower phase was then dialysed against a Tris/EDTA buffer and the plasmidwas finally isolated in this way.

Ribeiro et al. (Biotechnol. Bioeng. May 20, 2002;78(4):376-84)furthermore describe an aqueous 2-phase system with PEG as the polymercomponent and dipotassium hydrogen phosphate (K₂HPO₄) as the saltcomponent. The isolation of the plasmid pCF1-CFTR from E. coli DH5a isreported. For this, the cells were first broken down by means ofalkaline lysis (lysis buffer containing NaOH and SDS) and the lysate wasthen neutralized with 3 M sodium acetate. The cell debris, proteins andgenomic DNA (gDNA) were subsequently removed by centrifugation of thebatch. The clarified lysate was employed in the abovementioned aqueous2-phase system. However, this process has the disadvantages that it isvery expensive in terms of apparatus and time. Last but not least, thereason for the time consumed is that a lysis of the biomass is firstcarried out in a separate batch in this system and more than onereaction vessel is therefore necessary for such a process.

The present invention is intended to overcome the disadvantages of theprocesses known from the prior art, and in particular the object of theinvention is simplification and shortening in the time of the processesknown from the prior art for isolation of plasmid DNA from biomass. Inaddition, the object of the present invention is to provide a processwhich can be carried out not only on a laboratory scale but also on anindustrial scale and with which e.g. plasmid DNA for clinical use ingene therapy or genetic vaccination can be prepared. A further object ofthe present invention is to provide a process which can easily beautomated.

The object is achieved according to the invention by a process forisolation of plasmid DNA from biomass by means of an aqueous 2-phasesystem having a polymer component and a salt component, characterized inthat the resuspension of the biomass employed, the alkaline lysis of thebiomass, the neutralization of the alkaline lysis batch and theseparation of the plasmid DNA from the contaminants (such as e.g. celldebris, RNA and gDNA) are carried out in a single reaction vessel(one-pot process). This is rendered possible according to the inventionin that the neutralization of the alkaline lysis batch is carried out inone and the same container by addition of potassium phosphate and onecomponent of the aqueous 2-phase system is therefore already present,and in that the second component of the aqueous 2-phase system is a PEGhaving a molecular weight of the mathematical average of about 600 g/molto 1,000 g/mol, but is preferably formed by a mixture of PEG 600 and PEG1000.

The process according to the invention comprises the following steps:

-   -   a) resuspension of the biomass,    -   b) addition of lysis buffer, incubation for a sufficiently long        period of time for lysis of the biomass,    -   c) addition of the salt component, incubation for a sufficiently        long period of time,    -   d) addition of the polymer component, thorough mixing of the        solution, awaiting of the formation of phases.

Because of the simplicity due to the one-pot process, the processaccording to the invention is advantageously an automatable process forisolation of plasmid DNA. There is an ever increasing demand forautomatability of such processes on a relatively small scale, e.g. forrapid identification of clones. However, such an automatability can berealized only with difficulty with the known 2-phase systems. Only withthe process according to the invention can such an automation be easilyachieved.

The process claimed can also advantageously be employed for theproduction of plasmid DNA on an industrial scale. The reason for thislies in the simplicity, the low expenditure on apparatus and the use ofnon-toxic and inexpensive components.

One of the features of this process which are essential to the inventionis that the resuspension of the biomass employed, the alkaline lysis ofthe biomass, the neutralization of the lysis batch and the separation ofthe plasmid DNA from the contaminants take place in an aqueous 2-phasesystem successively in a single container, that is to say as a one-potprocess, without there being an intermediate centrifugation step withsubsequent separating off of a precipitate. The process according to theinvention is greatly simplified by this means, is significantly lessexpensive in terms of apparatus, and is greatly shorter in time comparedwith the processes known from the prior art.

The process according to the invention uses a 2-phase system of apolymer component and a salt component. This has the advantage that thesalts used are relatively inexpensive compared with a second polymercomponent (e.g. dextran) in a 2-phase system having two differentpolymer components.

In the case of the present invention, the term “plasmid DNA” alsoincludes, in addition to plasmids, cosmids as well as phasmids, and alsoeukaryotic vectors, such as e.g. yeast vectors, such as, for example,yeast artificial chromosomes (YACs) and similar structures. In thecontext of the present invention, the term “biomass” includes all lifeforms which are capable of carrying and multiplying or passing on totheir descendants this plasmid DNA, such as e.g. bacteria, yeasts etc.

The biomass which carries the plasmid DNA is obtained in this context bycentrifugation of an appropriate incubation batch or any other suitablemethod for concentrating the biomass. The person skilled in the art isfamiliar with such methods and how to carry them out. The resuspensionof the biomass, e.g. of the bacteria pellet, and the alkaline lysis ofthe biomass subsequently likewise proceed in a manner and way known tothe person skilled in the art, e.g. by means of the commerciallyobtainable QIAGEN Plasmid Kits and the resuspension buffer P1 and lysisbuffer P2 contained therein (QIAGEN, Hilden, Germany) or other suitablebuffers, in a container which seems suitable to the person skilled inthe art. Up the this point the procedure is conventionally in accordancewith the particular known instructions. The lysis buffer preferablycontains sodium dodecyl sulphate (SDS) as one of the components.

Advantageously and surprisingly, the lysis of the biomass and the lysateduring and after addition of the salt component can furthermore also becarried out under agitation of the lysis batch or lysate, such asvigorous shaking and/or stirring or the like. The processes known fromthe prior art have the disadvantage that the gDNA fragments necessarilyformed by shearing forces during such a treatment of the lysis batch orlysate cannot be separated to a satisfactory degree from the plasmid DNAto be isolated. Such a treatment of the lysis batch or lysate istherefore usually avoided by the person skilled in the art. In theaqueous 2-phase system according to the invention, however, the gDNAfragments formed in this way are also separated off from the plasmidDNA. This results on the one hand in a lower susceptibility of thesystem towards contamination of the plasmid DNA by gDNA, and furthermoreagitation of the lysis batch, such as e.g. vigorous stirring, shaking orthe like, additionally advantageously increases the release of theplasmid DNA from the biomass, and the agitation of the lysate, such ase.g. vigorous stirring, shaking or the like, during and/or afteraddition of the salt component according to the invention additionallyadvantageously increases the release of the plasmid DNA from theprecipitate agglomerates which form, which leads to a significantincrease in the yield of plasmid DNA. In a preferred embodiment, thegDNA sheared by agitation of the lysis batch or lysate is removed fromthe plasmid DNA to the extent of >90%, particularly preferably to theextent of >95% and very particularly preferably to the extent of >99%,compared with the otherwise conventional potassium acetate precipitationafter the lysis.

The substances which are conventional for the person skilled in the art,such as e.g. potassium acetate, are not resorted to for neutralizationof the batch of the alkaline lysis and for precipitation of theproteins. According to the invention, the alkaline lysis batch isadvantageously neutralized with the salt component according to theinvention. The salt component in the context of the invention ispotassium phosphate, by which is understood, according to the invention,tripotassium phosphate (K₃PO₄), dipotassium hydrogen phosphate (K₂HPO₄)and/or potassium dihydrogen phosphate (KH₂PO₄). Preferably, exclusivelyone of these or a mixture of these potassium phosphates is added to thealkaline lysis batch. K₂HPO₄ and/or KH₂PO₄ is particularly preferablyused. If SDS is used in the lysis buffer, the process according to theinvention furthermore renders possible the precipitation of proteins outof the lysis batch in the form of potassium dodecyl sulphate(PDS)-protein complexes.

According to the invention, the salt component is employed such that a2-phase system forms together with one of the polymer componentcompositions and concentrations according to the invention, but theconcentration of the salt component at which the plasmid DNA changesfrom the lower phase (salt phase), in which it is at lowerconcentrations, into the upper phase (polymer phase) not being exceeded,and contaminants, such as RNA and gDNA, remaining in the upper phase orin the interphase. It is well-known that 2-phase systems occur only whenlimiting concentrations are exceeded. The course of such limitingconcentrations can be plotted in a phase diagram. This is easy toachieve for the person skilled in the art.

In a preferred embodiment of the process according to the invention, thedepletion of the RNA contaminants from the plasmid DNA is >80%, comparedwith the potassium acetate precipitation conventionally used after lysisof the biomass, the depletion in the RNA contaminants is particularlypreferably >85% and the depletion in the RNA contaminants is veryparticularly preferably >90%.

For the process according to the invention, the potassium phosphate ispreferably added in the form of a buffer. In this context, the bufferparticularly preferably contains a mixture of K₂HPO₄ and KH₂PO₄. Thebuffers according to the invention are employed with a pH in the rangeof from pH 5.8 to pH 8.5 and preferably with a pH in the range of frompH 6.5 to pH 8. For example, a composition of 3.83 M K₂HPO₄ and 2.45 MKH₂PO₄ (a pH of approx. 7 results) can particularly preferably be usedin the process according to the invention. In this context, K₂HPO₄ andKH₂PO₄ are employed in a total concentration, based on the 2-phasesystem, of 5-30% (w/w), preferably in a total concentration of 10-25%(w/w) and particularly preferably in a total concentration of 20% (w/w).The potassium phosphate is usually added in a temperature range ofbetween ice-cooled and room temperature. Room temperature in the contextof the present invention designates a temperature range of from 18 to25° C. An ice-cooled phosphate buffer is preferably employed in theprocess according to the invention. A long incubation time isadvantageously not necessary after addition of the potassium phosphate,a thorough mixing of the solution which is as complete and uniform aspossible, after the addition, is decisive. The incubation time isusually about 5 to 15 minutes. Preferably, as mentioned above, the batchis agitated, such as e.g. subjected to vigorous shaking, stirring or thelike, during and/or after the addition of the salt component.

The polymer component in the context of the invention is PEG. A furtheressential feature of the process according to the invention is thatpolyethylene glycol having a molecular weight of the mathematicalaverage of 600 to 1,000 g/mol, preferably of the mathematical average of700-900 g/mol and particularly preferably of the mathematical average of750-880 g/mol is employed as one of the two components of the 2-phasesystem. In the present invention, the PEG employed preferably comprisesa mixture of polyethylene glycol having an average molecular weight of600 g/mol (PEG 600) and polyethylene glycol having an average molecularweight of 1,000 g/mol (PEG 1000). Both PEGs are commercially obtainable(e.g. Fluka, Buchs, Switzerland). In this context, the ready-to-use PEGmixture comprises 30-50% (w/w) PEG 600 and 50-70% (w/w) PEG 1000,preferably 33-45% (w/w) PEG 600 and 55-67% (w/w) PEG 1000, particularlypreferably 36-40% (w/w) PEG 600 and 60-64% (w/w) PEG 1000 and veryparticularly preferably 38% (w/w) PEG 600 and 62% (w/w) PEG 1000.

The concentration of the PEG in the aqueous 2-phase system according tothe invention is chosen such that two phases are formed with the saltcomponent, but the PEG concentration at which the plasmid DNAs changefrom the lower phase, in which they are at lower concentrations, intothe upper phase not being exceeded. Preferably, however, the PEG contentis at least 10% (w/w), and the upper limit is determined by theconcentration of PEG at which the plasmid DNAs change from the lowerphase, in which they are at lower concentrations, into the upper phase.After the addition of PEG, the solution should preferably have atemperature of from 10 to 50° C., particularly preferably a temperatureof from 15 to 40° C. After formation of the phases, which takes someminutes to hours, depending on the volume of the batch, the plasmid DNAis in the salt-containing lower phase. The formation of the phases canoptionally be accelerated by centrifugation of the batch, as a result ofwhich there is advantageously a further shortening in the time of theprocess according to the invention. The conditions under which such acentrifugation step is carried out are familiar to the person skilled inthe art.

Aqueous 2-phase systems have the advantage over phase systems whichoperate on the basis of matrices or other solid phases that they have afar higher capacity for the plasmid DNA to be purified, which is limitedin practice only by the solubility in the phases. Furthermore, thedimensions of the process can be chosen virtually as desired because ofthe extremely simple apparatus. However, only with the simplificationsclaimed here can both an automation and, independently of this, aproduction on an industrial scale, for example for the production ofmore than 2 g plasmid DNA per lysis batch, be easily achieved. Theplasmid DNA can also advantageously be freed from proteins, RNA and gDNAto a very high degree with the present invention. Plasmid DNA which isisolated by the process according to the invention can be employedwithout problems in gene therapy or genetic vaccination after a furtherpurification step (for example via QIAGEN-Resin, QIAGEN, Hilden,Germany). Thus, large amounts of highly pure plasmid DNA canadvantageously be produced with a very low expenditure on apparatususing non-toxic substances and with comparatively low costs beingincurred. With regard to this it may be mentioned that the substancesemployed in the 2-phase system according to the invention, however, areacceptable e.g. compared with other isolation methods, such as e.g. CsCldensity gradient centrifugation or phenol extraction, and can be removedcompletely and easily from the purified plasmid DNA.

One of the great advantages of the process according to the invention isthat, surprisingly, there is a depletion in the open circular plasmidDNA (ocDNA) from the plasmid DNA to be isolated. The plasmid DNAisolated with the process according to the invention has, compared withplasmid DNA which has been isolated with processes known from the priorart, a lower content of the ocDNA contaminants in comparison with thepreferred supercoiled form (scDNA). This process thus shows aselectivity for separation of plasmid topoisomers, that is to say ofocDNA and scDNA. Precisely this separation of the plasmid topoisomersand the depletion of the gDNA already mentioned are of great importancein particular in an isolation of plasmid DNA on an industrial scale andduring subsequent clinical use of the plasmid DNA in some applications,and are not to be achieved to a satisfactory degree with the processesto date for isolation of plasmid DNA.

In a further embodiment of the process according to the invention, thelower phase containing the plasmid DNA forming in step d) is separatedfrom the upper phase containing the contaminants. The lower phase isthen set up again for the formation of a 2-phase system by addition ofthe salt component in the form which results in a concentration of thesalt component according to step c), and then by addition of the polymercomponent according to step d). In this repetition of the last part ofthe process according to the invention, after the addition of thepolymer component the mixture is mixed thoroughly again and theformation of phases is awaited. Here also, the plasmid DNA is in thelower phase. This step can optionally be carried out once or in a numberof times which seem appropriate to the person skilled in the art.Carrying out this optional step leads to a repeated purification of theplasmid DNA and therefore to a further depletion of contaminants, suchas e.g. RNA, from the plasmid DNA. The additional purification step(s)comprise(s) the following steps:

-   -   e) separation of the lower phase forming in step d) from the        upper phase formed in step d) and containing the contaminants,    -   f) addition of the salt component in the form which results in a        concentration of the salt component according to step c),    -   g) renewed addition of the polymer component according to step        d),    -   h) thorough mixing of the solution, awaiting of the formation of        phases.

After the process according to the invention, the plasmid DNA must stillbe obtained from the lower phase formed in step d). The isolation anddesalination of the plasmid DNA from the lower phase formed in step d)are conventionally carried out by ultrafiltration/diafiltration.However, any further method of isolation and/or desalination of theplasmid DNA from the lower phase which seems appropriate to the personskilled in the art may also be used in the context of the presentinvention.

In an alternative embodiment, instead of the biomass, a clarified lysatewhich contains the plasmid DNA to be isolated can also be employed asthe starting material in the process according to the invention. In thisembodiment of the invention, the lysis step is of course omitted, or aclarification of the lysate additionally takes place after the lysisstep. All the methods which seem suitable to the person skilled in theart can be used for the preparation of a clarified lysate. Aconventional method in the prior art is clarification of the lysate byprecipitation with potassium acetate and subsequent centrifugation ofthe mixture to separate off the precipitate formed. For this case,potassium phosphate corresponding to the above statements is first addedto the clarified lysate, and PEG is then added according to theinvention. All further steps are identical to the process according tothe invention in which biomass is employed as the starting point. If theprecipitation step described above for clarification of the lysate iscarried out with potassium phosphate, the PEG is added, as described,directly to the clarified lysate. All further steps are identical to theprocess according to the invention in which biomass is employed as thestarting point. The experimental conditions which are required for aprecipitation reaction as described above, both if potassium phosphateis used and if potassium acetate is used, are well-known to the personskilled in the art.

EXPLANATION OF THE FIGURE

FIG. 1 shows the results of the isolation of pCMVβ from E. coli DH5a bythe process according to the invention (see Example 1) in the form of agel electrophoresis (0.8% agarose). A length standard can be seen intrack 1. Track 2 shows the untreated upper phase, in which there is amassive amount of RNA. No plasmid DNA could be detected in the upperphase. Track 3 shows the untreated lower phase, in which the plasmid ishighly concentrated (wide, blurred band), no RNA could be detected here.Track 4 shows the lower phase after desalination by means ofultrafiltration. Here also, only plasmid DNA can be detected, and theplasmid is mostly in the desired supercoiled form (scDNA), traces ofopen circular plasmid DNA (ocDNA) could also be detected. Track 5 showsa pCMVβ standard (18 μg/ml) and track 6 shows a gDNA standard (23μg/ml).

EXAMPLE 1 Obtaining of pCMVβ from E. coli DH5a

For isolation of the plasmid pCMVβ, 0.6 g biomass from an overnightculture of E. coli DH5a (11 LB medium: 5 g/l yeast extract, 10 g/ltryptone, 85.6 mM NaCl) was obtained by centrifugation (5,000×g, 10 min,4° C.). 6.9 ml resuspension buffer P1 (QIAGEN, Hilden, Germany) wereadded and the bacteria pellet was resuspended by vigorous shaking atroom temperature. When the resuspension was complete, 7.5 ml lysisbuffer P2 (QIAGEN, Hilden, Germany; room temperature) were added and thebatch was mixed by inverting the vessel several times. After 5 min,K₂HPO₄/KH₂PO₄ buffer (2.5 g KH₂PO₄, 5 g K₂HPO₄, 7.5 ml H₂O; pH 7;ice-cooled) was added to the entire batch, the vessel was cautiouslyinverted and the batch was incubated on ice for 10 min. The entire batchwas subsequently warmed to room temperature, PEG (2.137 g PEG 600, 3.488g PEG 1000, 1.875 ml H₂O) was added and the batch was cautiously mixed.The phase separation took a few minutes.

No gDNA and no RNA could be detected in the lower phase by means of gelelectrophoresis. The band of the plasmid DNA looks untypically wide andblurred in FIG. 1 (track 3) because of the high salt concentration inthe lower-phase. Desalination of the lower phase by means ofultrafiltration (track 4) led to a correspondingly distinct band whichvirtually corresponds to the plasmid standard (track 5). Genomic DNA andproteins are to be found in the potassium dodecyl sulphate (PDS)flocculate and in the interphase.

EXAMPLE 2 Obtaining of pCMVβ from E. coli DH5a (20% PEG; 12% Phosphate)

For isolation of the plasmid pCMVβ, a 10% (w/w) biomass suspension (E.coli DH5a) in resuspension buffer P1 (QIAGEN, Hilden, Germany) wasprepared by vigorous shaking at room temperature. When the resuspensionwas complete, 10 g of this batch were mixed with 10 ml lysis buffer P2(QIAGEN, Hilden, Germany; room temperature) and the vessel was invertedseveral times. After 5 min, K₂HPO₄/KH₂PO₄ buffer (2 g KH₂PO₄; 4 gK₂HPO₄; 6 ml H₂O; pH 7.4; ice-cooled) was added to the entire batch andthe batch was incubated on an overhead shaking machine at roomtemperature for 10 min. The entire batch was subsequently warmed to roomtemperature, PEG (3.8 g PEG 600; 6.2 g PEG 1000; 8.003 ml H₂O) was addedand the batch was mixed on an overhead shaking machine at roomtemperature for a further 10 min. The phase separation was acceleratedby centrifugation at 1,300×g for 5 min. The resulting lower phase had avolume of about 15 ml.

Analysis was carried out by means of HPLC (HP1090, Agilent, Waldbronn,Germany) and source 15PHE 4.6/100 (Amersham Bioscience, Freiburg,Germany) employing an (NH₄)₂SO₄ gradient. Detection was by absorptionmeasurement at 260 nm. The following values were determined for thelower phase of the 2-phase system: 44 μg/ml plasmid DNA and 123 μg/mlRNA.

These values were compared with the values from a clarified lysate. Forpreparation of this clarified lysate, 1 ml of the 10% biomass suspensioninitially described was mixed with 1 ml lysis buffer P2 (QIAGEN, Hilden,Germany; room temperature) by inverting the vessel several times and thebatch was incubated at room temperature for 5 min. 1 ml neutralizationbuffer P3 (QIAGEN, Hilden, Germany; ice-cooled) was then added and thebatch was incubated on an overhead shaking machine at room temperaturefor 10 min. After centrifugation (5 min, 1,300×g, room temperature), theplasmid concentration and the RNA concentration in the supernatant(clarified lysate) were measured by means of HPLC. The biomass employedcontained in the clarified lysate 710 μg plasmid DNA per g biomass and11 mg RNA per g biomass.

Based on the starting values of the biomass in the clarified lysate, a93% plasmid yield and an 83% RNA depletion result for the 2-phasesystem.

EXAMPLE 3 Obtaining of pCMVβ from E. coli DH5a (13% PEG; 16% Phosphate)

For isolation of the plasmid pCMVβ, a 10% (w/w) biomass suspension (E.coli DH5a) in resuspension buffer P1 (QIAGEN, Hilden, Germany) wasprepared by vigorous shaking at room temperature. When the resuspensionwas complete, 10 g of this batch were mixed with 10 ml lysis buffer P2(QIAGEN, Hilden, Germany; room temperature) and the vessel was invertedseveral times. After 5 min, K₂HPO₄/KH₂PO₄ buffer (2.666 g KH₂PO₄; 5.333g K₂HPO₄; 8 ml H₂O; pH 7.4; ice-cooled) was added to the entire batchand the batch was incubated on an overhead shaking machine at roomtemperature for 10 min. The entire batch was subsequently warmed to roomtemperature, PEG (2.471 g PEG 600; 4.032 g PEG 1000; 7.498 ml H₂O) wasadded and the batch was mixed on an overhead shaking machine at roomtemperature for a further 10 min. The phase separation was acceleratedby centrifugation at 1,300×g for 5 min. The resulting lower phase had avolume of about 25 ml.

Analysis was carried out by means of HPLC (HP1090, Agilent, Waldbronn,Germany) and source 15PHE 4.6/100 (Amersham Bioscience, Freiburg,Germany) employing an (NH₄)₂SO₄ gradient. Detection was by absorptionmeasurement at 260 nm. The following values were determined for thelower phase of the 2-phase system: 28.4 μg/ml plasmid DNA and 187 μg/mlRNA.

These values were compared with the values from a clarified lysate. Forpreparation of this clarified lysate, 1 ml of the 10% biomass suspensioninitially described was mixed with 1 ml lysis buffer P2 (QIAGEN, Hilden,Germany; room temperature) by inverting the vessel several times and thebatch was incubated at room temperature for 5 min. 1 ml neutralizationbuffer P3 (QIAGEN, Hilden, Germany; ice-cooled) was then added and thebatch was incubated on an overhead shaking machine at room temperaturefor 10 min. After centrifugation (5 min, 1,300×g, room temperature), theplasmid concentration and the RNA concentration in the supernatant(clarified lysate) were measured by means of HPLC. The biomass employedcontained in the clarified lysate 710 μg plasmid DNA per g biomass and11 mg RNA per g biomass.

Based on the starting values of the biomass in the clarified lysate,a >99% plasmid yield and a 58% RNA depletion result for the 2-phasesystem.

EXAMPLE 4 Obtaining of pCMVβ from E. coli DH5a (30% PEG, 10% Phosphate)

For isolation of the plasmid pCMVβ, a 10% (w/w) biomass suspension (E.coli DH5a) in resuspension buffer P1 (QIAGEN, Hilden, Germany) wasprepared by vigorous shaking at room temperature. When the resuspensionwas complete, 10 g of this batch were mixed with 10 ml lysis buffer P2(QIAGEN, Hilden, Germany; room temperature) and the vessel was invertedseveral times. After 5 min, K₂HPO₄/KH₂PO₄ buffer (1.666 g KH₂PO₄; 3.333g K₂HPO₄; 5 ml H₂O; pH 7.4; ice-cooled) was added to the entire batchand the batch was incubated on an overhead shaking machine at roomtemperature for 10 min. The entire batch was subsequently warmed to roomtemperature, PEG (5.7 g PEG 600; 9.3 g PEG 1000; 5 ml H₂O) was added andthe batch was temperature-controlled in a water-bath at 40° C for 15 minand with inversion of the vessel several times. The phase separation wasaccelerated by centrifugation at 1,300×g for 5 min. The resulting lowerphase had a volume of about 11 ml.

Analysis was carried out by means of HPLC (HP1090, Agilent, Waldbronn,Germany) and source 15PHE 4.6/100 (Amersham Bioscience, Freiburg,Germany) employing an (NH₄)₂SO₄ gradient. Detection was by absorptionmeasurement at 260 nm. The following values were determined for thelower phase: 61.8 μg/ml plasmid DNA and 173 μg/ml RNA.

These values were compared with the values from a clarified lysate. Forpreparation of this clarified lysate, 1 ml of the 10% biomass suspensioninitially described was mixed with 1 ml lysis buffer P2 (QIAGEN, Hilden,Germany; room temperature) by inverting the vessel several times and thebatch was incubated at room temperature for 5 min. 1 ml neutralizationbuffer P3 (QIAGEN, Hilden, Germany; ice-cooled) was then added and thebatch was incubated on an overhead shaking machine at room temperaturefor 10 min. After centrifugation (5 min, 1,300×g, room temperature), theplasmid concentration and the RNA concentration in the supernatant(clarified lysate) were measured by means of HPLC. The biomass employedcontained in the clarified lysate 710 μg plasmid DNA per g biomass and11 mg RNA per g biomass.

Based on the starting values of the biomass in the clarified lysate, a96% plasmid yield and an 83% RNA depletion result for the 2-phasesystem.

EXAMPLE 5 Depletion of gDNA

A 20% biomass suspension was prepared by weighing out 4 g resuspensionbuffer P1 (QIAGEN, Hilden, Germany) and 1 g biomass (E. coli DH5a,containing no plasmid). This was broken down with ultrasound (BransonSonifier 250, with converter type 102, settings: cycle 40%; intensity 4,2 times 3 min) on ice. The gDNA was thereby released from the cell andsubjected to severe shearing forces and fragmented. The batch was thencentrifuged for 10 min at 5,000×g and 4° C. 300 μl lysis buffer P2(QIAGEN, Hilden, Germany; room temperature) were then added to 300 μg ofthe supernatant and the batch was mixed. After 5 min, the entire batchwas mixed with K₂HPO₄/KH₂PO₄ buffer (100 μg KH₂PO₄; 200 μg K₂HPO₄, 300μl H₂O; pH 7.4; ice-cooled) and the batch was incubated on an overheadshaking machine at room temperature for 10 min. The entire batch wassubsequently warmed to room temperature, PEG (85.5 μg PEG 600; 139.5 μgPEG 1000; 75 μl H₂O) was added and the batch was mixed on an overheadshaking machine at room temperature for a further 10 min. The phaseseparation was accelerated by centrifugation at 1,300×g for 5 min. Thelower phase had a volume of about 0.75 ml.

Analysis was carried out by means of HPLC (HP1090, Agilent, Waldbronn,Germany) and source 15PHE 4.6/100 (Amersham Bioscience, Freiburg,Germany) employing an (NH₄)₂SO₄ gradient. Detection was by absorptionmeasurement at 260 nm. The gDNA concentration in the supernatant of thebreaking down operation (centrifugation after ultrasound treatment) wasdetermined as 433 μg gDNA/ml. In the lower phase of the 2-phase system,a residual concentration of 1.7 μg gDNA/ml was determined by means ofHPLC, which corresponds to a depletion of >99% of severely sheared gDNA.

1-28. (canceled)
 29. A method for isolating plasmid DNA from biomass,said method comprising an aqueous 2-phase system having a polymercomponent and a salt component, said method comprising the steps of: (a)suspending the biomass, (b) adding lysis buffer and incubating for asufficient period of time for lysis of the biomass, (c) adding a saltcomponent and incubating for a sufficient period of time to neutralizethe lysis buffer, (d) adding a polymer component and thoroughly mixingthe solution, and (e) awaiting the formation of an upper phasecontaining contaminants and a lower phase containing the plasmid DNA,wherein the suspension of the biomass, the addition of the lysis buffer,salt component and polymer component are all carried out in a singlereaction vessel.
 30. The method according to claim 29, whereinpolyethylene glycol (PEG) is used as the polymer component of theaqueous 2-phase system.
 31. The method according to claim 30, whereinthe PEG employed has an average molecular weight of 600 to 1,000 g/mol.32. The method according to claim 31 wherein the PEG employed is amixture of 30-50% (w/w) PEG having an average molecular weight of 600g/mol (PEG 600) and 50-70% (w/w) PEG having an average molecular weightof 1,000 g/mol (PEG 1000).
 33. The method according to claim 30, whereinthe concentration of the PEG is chosen such that it is at least so highthat two phases are formed with the salt component of the 2-phasesystem, and is at most so high that the concentration at which theplasmid DNA changes from the lower phase into the upper phase is notexceeded and contaminants, such as RNA and gDNA, remain in the upperphase or in the interphase.
 34. The method according to claim 33,wherein the content of PEG is at least 10% (w/w) and is at most so highthat the concentration at which the plasmid DNA changes from the lowerphase into the upper phase is not exceeded and contaminants, such as RNAand gDNA, remain in the upper phase or in the interphase.
 35. The methodaccording to claim 29, wherein tripotassium phosphate (K₃PO₄),dipotassium hydrogen phosphate (K₂HPO₄ and/or potassium dihydrogenphosphate (KH₂PO₄) are used as the salt component.
 36. The methodaccording to claim 35, wherein the salt component is added in the formof a buffer solution.
 37. The method according to claim 36, wherein thebuffer solution is K₂HPO₄/KH₂PO₄ buffer.
 38. The method according toclaim 35, wherein the concentration of the salt component is chosen suchthat it is at least so high that two phases are formed with the polymercomponent of the 2-phase system, and is at most so high that theconcentration of the salt component at which the plasmid DNA changesfrom the lower phase into the upper phase is not exceeded andcontaminants, such as RNA and gDNA, remain in the upper phase or in theinterphase.
 39. The method according to claim 38, wherein K₂HPO₄ and/orKH₂PO₄ are employed in a total concentration of 5-30% (w/w).
 40. Themethod according to either of claims 36 or 39, wherein the buffer systemof the salt component has a pH in the range of from pH 5.8 to pH 8.5.41. The method according to claim 29, wherein the plasmid DNA to beisolated includes plasmids, cosmids and phasmids as well as eukaryoticvectors.
 42. The method according to claim 41, wherein the eukaryoticvectors are yeast vectors.
 43. The method according to claim 29, whereinthe lysis buffer is an alkaline lysis buffer containing sodium dodecylsulphate (SDS).
 44. The method according to claim 43, wherein thereaction vessel is agitated to assist in lsying the biomass.
 45. Themethod according to claim 29, wherein the biomass is a clarified lysate.46. The method according to claim 29, wherein the lower phase forming instep d) from claim 1 is additionally purified from contaminants.
 47. Themethod according to claim 29, further comprising the additionalpurification steps: (f) separating the lower phase forming in step (e)from the upper phase formed in step (e), (g) adding the salt componentto the separated lower phase from step (f), (h) adding the polymercomponent, and (i) mixing of the solution and awaiting of the formationof phases.
 48. The method according to either claim 29 or 47, whereinthe plasmid DNA in the lower phase is further isolated and desalinatedby ultrafiltration/diafiltration.
 49. The method according to claim 29,wherein the formation of the phases in step (e) is accelerated bycentrifugation.
 50. The method according to claim 29, wherein themixture from step (c) from claim 1 is agitated during and/or after theaddition of the salt component.
 51. The method according to claim 29,wherein the method is automated.